# US 11,078,493 B2 · Site-specific DNA recombination

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- Number: US 11,078,493 B2
- Document ID: US11078493B2
- Status: Granted
- Kind: B2
- Application number: 15/629,334
- Filed: 2017-06-21
- Published: 2017-12-21
- Granted: 2021-08-03
- Earliest claimed: 2016-06-21
- Assignees: Nanyang Technological University
- Applicants: Nanyang Technological University
- Inventors: Harshyaa Makhija; Peter Droge
- Web page: https://patentgrep.com/patent/US11078493B2

## CPC

- C12N 15/85: for animal cells
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 15/907: {in mammalian cells}
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2800/107: for mammalian
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention provides a method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the target genomic DNA sequence comprises a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, as well as a kit for use in said method. Also provided is a method of generating a circular DNA construct essentially consisting of a DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3.

## Claims

### Claim 1

A method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the target genomic DNA sequence comprises a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, the method comprising the steps of:
(i) providing a bacterial plasmid comprising the DNA sequence of interest flanked on a first end by a first nucleotide sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and on a second end by a second nucleotide sequence comprising a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2, wherein the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 flanking the DNA sequence of interest are arranged in a directly repeated orientation;
(ii) subjecting the bacterial plasmid to conditions that allow intramolecular recombination between the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 in the presence of a phage lambda integrase, to obtain a first circular DNA construct essentially consisting of the DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3 that does not contain bacterial sequences, except the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3, and a second circular DNA construct comprising the remainder sequence of the bacterial plasmid and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:4;
(iii) linearizing the second circular DNA construct by means of an endonuclease activity;
(iv) isolating the first circular DNA construct comprising the DNA sequence of interest;
(v) introducing the first circular DNA construct into the host cell, said cell further comprising a phage lambda integrase; and
(vi) subjecting the host cell to conditions that allow integration of the DNA sequence of interest into the target genomic DNA sequence of the host cell, wherein said integration is mediated by the phage lambda integrase.

### Claim 2 (depends on 1)

The method of claim 1, wherein the phage lambda integrase is selected from the group consisting of wild-type Int (SEQ ID NO:5), Int-h (E174K; SEQ ID NO:6), Int-h/218 (E174K/E218K; SEQ ID NO:7) and polypeptide sequences that are at least 80% homologous thereto.

### Claim 3 (depends on 2)

The method of claim 2, wherein the phage lambda integrase is N- or C-terminally fused to a nuclear localizing sequence (NLS; SEQ ID NO:9 or a nucleotide sequence that is at least 80% homologous thereto).

### Claim 4 (depends on 1)

The method of claim 1, wherein the phage lambda integrase and the first circular DNA construct comprising the DNA sequence of interest are co-introduced or independently introduced into the host cell by DNA transfection, viral transduction, biolistic technology, ultrasound, nanoparticles or microinjection.

### Claim 5 (depends on 1)

The method of claim 1, wherein the intramolecular recombination of step (ii) is carried out in the presence of an IHF variant.

### Claim 6 (depends on 1)

The method of claim 1, wherein the DNA sequence of interest comprises one or more genes.

### Claim 7 (depends on 6)

The method of claim 6, wherein at least one of the one or more genes is operably linked to expression control sequence(s).

### Claim 8 (depends on 6)

The method of claim 6, wherein the DNA sequence of interest comprises a selection marker gene.

### Claim 9 (depends on 1)

The method of claim 1, wherein the target genomic DNA sequence comprising the nucleotide sequence as set forth in SEQ ID NO:1 is located in Long Interspersed Elements-1 (LINE-1) elements of the genome of the host cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the host cell is a human cell.

### Claim 11

A method of generating a circular DNA construct essentially consisting of a DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3, wherein the circular DNA construct comprising the DNA sequence of interest does not contain bacterial sequences, except the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3, and wherein the circular DNA construct comprising the DNA sequence of interest allows integration of the DNA sequence of interest into a target genomic DNA sequence of a host cell by intermolecular recombination mediated by phage lambda integrase, said target genomic sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, the method comprising the steps of:
(i) providing a bacterial plasmid comprising the DNA sequence of interest flanked on a first end by a first nucleotide sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and on a second end by a second nucleotide sequence comprising a sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2, wherein the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 flanking the DNA sequence of interest are arranged in a directly repeated orientation;
(ii) subjecting the bacterial plasmid to conditions that allow intramolecular recombination between the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 in the presence of a phage lambda integrase, to obtain a first circular DNA construct essentially consisting of the DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3 that does not contain bacterial sequences, except the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3, and a second circular DNA construct comprising the remainder sequence of the bacterial plasmid and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:4;
(iii) linearizing the second circular DNA construct by means of an endonuclease activity; and
(iv) isolating the first circular DNA construct comprising the DNA sequence of interest.

### Claim 12 (depends on 11)

The method of claim 11, wherein the phage lambda integrase is selected from the group consisting of wild-type Int (SEQ ID NO:5), Int-h (E174K; SEQ ID NO:6), Int-h/218 (E174K/E218K; SEQ ID NO:7), and polypeptide sequences that are at least 80% homologous thereto.

### Claim 13 (depends on 11)

The method of claim 11, wherein the intramolecular recombination of step (ii) is carried out in the presence of an THF variant.

### Claim 14 (depends on 11)

The method of claim 11, wherein the DNA sequence of interest comprises one or more genes.

### Claim 15 (depends on 14)

The method of claim 14, wherein at least one of the one or more genes is operably linked to expression control sequence(s).

### Claim 16 (depends on 14)

The method of claim 14, wherein the DNA sequence of interest comprises a selection marker gene.